Systems and methods for preparing and analyzing low volume analyte array elements

ABSTRACT

Serial and parallel dispensing tools that can deliver defined and controlled volumes of fluid to generate multi-element arrays of sample material on a substrate surface are provided. The substrates surfaces can be flat or geometrically altered to include wells of receiving material. Also provided are tools that allow the parallel development of a sample array. To this end, the tool can be understood as an assembly of vesicle elements, or pins, where each of the pins can include a narrow interior chamber suitable for holding nanoliter volumes of fluid. Each of the pins can fit inside a housing that forms an interior chamber. The interior chamber can be connected to a pressure source that will control the pressure within the interior chamber to regulate the flow of fluid within the interior chamber of the pins. The prepared sample arrays can then be passed to a plate assembly that disposes the sample arrays for analysis by mass spectrometry.

RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No.08/787,639 to Little et al., entitled SYSTEMS AND METHODS FOR PREPARINGAND ANALYZING LOW VOLUME ANALYTE ARRAY ELEMENTS, filed Jan. 23, 1997.The subject matter of U.S. application Ser. No. 08/787,639 isincorporated herein in its entirety.

FIELD OF THE INVENTION

[0002] The invention relates to systems and methods for preparing asample for analysis, and more specifically to systems and methods fordispensing low volumes of fluid material onto a substrate surface forgenerating an array of samples for diagnostic analysis.

BACKGROUND OF THE INVENTION

[0003] In recent years, developments in the field of life sciences haveproceeded at a breathtaking rate. Universities, hospitals and newlyformed companies have made groundbreaking scientific discoveries andadvances that promise to reshape the fields of medicine, agriculture,and environmental science. However, the success of these effortsdepends, in part, on the development of sophisticated laboratory toolsthat will automate and expedite the testing and analysis of biologicalsamples. Only upon the development of such tools can the benefits ofthese recent scientific discoveries be achieved fully.

[0004] At the forefront of these efforts to develop better analyticaltools is a push to expedite the analysis of complex biochemicalstructures. This is particularly true for human genomic DNA, which iscomprised of at least about one hundred thousand genes located on twentyfour chromosomes. Each gene codes for a specific protein, which fulfillsa specific biochemical function within a living cell. Changes in a DNAsequence are known as mutations and can result in proteins with alteredor in some cases even lost biochemical activities; this in turn cancause a genetic disease. More than 3,000 genetic diseases are currentlyknown. In addition, growing evidence indicates that certain DNAsequences may predispose an individual to any of a number of geneticdiseases, such as diabetes, arteriosclerosis, obesity, certainautoimmune diseases and cancer. Accordingly, the analysis of DNA is adifficult but worthy pursuit that promises to yield informationfundamental to the treatment of many life threatening diseases.

[0005] Unfortunately, the analysis of DNA is made particularlycumbersome due to size and the fact that genomic DNA includes bothcoding and non-coding sequences (e.g., exons and introns). As such,traditional techniques for analyzing chemical structures, such as themanual pipeting of source material to create samples for analysis, areof little value. To address the scale of the necessary analysis,scientist have developed parallel processing protocols for DNAdiagnostics.

[0006] For example, scientists have developed robotic devices thateliminate the need for manual pipeting and spotting by providing arobotic arm that carries at its proximal end a pin tool device thatincludes a matrix of pin elements. The individual pins of the matrix arespaced apart from each other to allow each pin be dipped within a wellof a microtiter plate. The robotic arm dips the pins into the wells ofthe microtiter plate thereby wetting each of the pin elements withsample material. The robotic arm then moves the pin tool device to aposition above a target surface and lowers the pin tool to the surfacecontacting the pins against the target to form a matrix of spotsthereon. Accordingly, the pin tool expedites the production of samplesby dispensing sample material in parallel.

[0007] Although this pin tool technique works well to expedite theproduction of sample arrays, it suffers from several drawbacks. Firstduring the spotting operation, the pin tool actually contacts thesurface of the substrate. Given that each pin tool requires a fine pointin order that a small spot size is printed onto the target, thecontinuous contact of the pin tool against the target surface will wearand deform the fine and delicate points of the pin tool. This leads toerrors which reduce accuracy and productivity.

[0008] An alternative technique developed by scientists employs chemicalattachment of sample material to the substrate surface. In oneparticular process, DNA is synthesized in situ on a substrate surface toproduce a set of spatially distinct and diverse chemical products. Suchtechniques are essentially photolithographic in that they combine solidphase chemistry, photolabile protecting groups and photo activatedlithography. Although these systems work well to generate arrays ofsample material, they are chemically intensive, time consuming, andexpensive.

[0009] It is further troubling that neither of the above techniquesprovide sufficient control over the volume of sample material that isdispensed onto the surface of the substrate. Consequently, error canarise from the failure of these techniques to provide sample arrays withwell controlled and accurately reproduced sample volumes. In an attemptto circumvent this problem, the preparation process will often dispensegenerous amounts of reagent materials. Although this can ensuresufficient sample volumes, it is wasteful of sample materials, which areoften expensive and of limited availability.

[0010] Even after the samples are prepared, scientists still mustconfront the need for sophisticated diagnostic methods to analyze theprepared samples. To this end, scientists employ several techniques foridentifying materials such as DNA. For example, nucleic acid sequencescan be identified by hybridization with a probe which is complementaryto the sequence to be identified. Typically, the nucleic acid fragmentis labeled with a sensitive reporter function that can be radioactive,fluorescent, or chemiluminescent. Although these techniques can workwell, they do suffer from certain drawbacks. Radioactive labels can behazardous and the signals they produce decay over time. Nonisotopic(e.g. fluorescent) labels suffer from a lack of sensitivity and fadingof the signal with high intensity lasers are employed during theidentification process. In addition, labeling is a laborious and timeconsuming error prone procedure.

[0011] Consequently, the process of preparing and analyzing arrays of abiochemical sample material is complex and error prone.

SUMMARY OF THE INVENTION

[0012] Accordingly, it is an object herein to provide improved systemsand methods for preparing arrays of sample material.

[0013] It is a further object to provide systems that allow for therapid production of sample arrays.

[0014] It is yet another object to provide systems and methods forpreparing arrays of sample material that are less expensive to employand that conserve reagent materials.

[0015] It is a further object to provide systems and methods forpreparing arrays of sample material that provide high reproducibility ofthe arrays generated.

[0016] Other objects of the apparatus and methods provided herein willbe apparent from the description also disclosed in the following.

[0017] Serial and parallel dispensing tools that can be employed togenerate multi-element arrays of sample material on a substrate surfaceare provided. The substrates surfaces can be flat or geometricallyaltered to include wells of receiving material. In one embodiment, atool that allows the parallel development of a sample array is provided.To this end, the tool can be understood as an assembly of vesicleelements, or pins, wherein each of the pins can include a narrowinterior chamber suitable for holding nano liter volumes of fluid. Eachof the pins can fit inside a housing that itself has in interiorchamber. The interior housing can be connected to a pressure source thatwill control the pressure within the interior housing chamber toregulate the flow of fluid through the interior chamber of the pins.This allows for the controlled dispensing of defined volumes of fluidfrom the vesicles. In an alternative embodiment, the invention providesa tool that includes a jet assembly that can include a capillary pinhaving an interior chamber, and a transducer element mounted to the pinand capable of driving fluid through the interior chamber of the pin toeject fluid from the pin. In this way, the tool can dispense a spot offluid to a substrate surface by spraying the fluid from the pin.Alternatively, the transducer can cause a drop of fluid to extend fromthe capillary so that fluid can be passed to the substrate by contactingthe drop to the surface of the substrate. Further, the tool can form anarray of sample material by dispensing sample material in a series ofsteps, while moving the pin to different locations above the substratesurface to form the sample array. In a further embodiment, the inventionthen passes the prepared sample arrays to a plate assembly that disposesthe sample arrays for analysis by mass spectrometry. To this end, a massspectrometer is provided that generates a set of spectra signal whichcan be understood as indicative of the composition of the samplematerial under analysis.

[0018] To this end a dispensing apparatus for dispensing defined volumesof fluid, including nano and sub-nano volumes of fluid, in chemical orbiological procedures onto the surface of a substrate is provided. Theapparatus provided herein can include a housing having a plurality ofsides and a bottom portion having formed therein a plurality ofapertures, the walls and bottom portion of the housing defining aninterior volume; one or more fluid transmitting vesicles, or pins,mounted within the apertures, having a nanovolume sized fluid holdingchamber for holding nanovolumes of fluid, the fluid holding chamberbeing disposed in fluid communication with the interior volume of thehousing, and a dispensing element that is in communication with theinterior volume of the housing for selectively dispensing nanovolumes offluid form the nanovolume sized fluid transmitting vesicles when thefluid is loaded with the fluid holding chambers of the vesicles. Asdescribed herein, this allows the dispensing element to dispensenanovolumes of the fluid onto the surface of the substrate when theapparatus is disposed over and in registration with the substrate.

[0019] In one embodiment the fluid transmitting vesicle has an openproximal end and a distal tip portion that extends beyond the housingbottom portion when mounted within the apertures. In this way the openproximal end can dispose the fluid holding chamber in fluidcommunication with the interior volume when mounted with the apertures.Optionally, the plurality of fluid transmitting vesicles are removablyand replaceably mounted within the apertures of the housing, oralternatively can include a glue seal for fixedly mounting the vesicleswithin the housing.

[0020] In one embodiment the fluid holding chamber includes a narrowbore dimensionally adapted for being filled with the fluid throughcapillary action, and can be sized to fill substantially completely withthe fluid through capillary action.

[0021] In one embodiment, the plurality of fluid transmitting vesiclescomprise an array of fluid delivering needles, which can be formed ofmetal, glass, silica, polymeric material, or any other suitablematerial.

[0022] In one embodiment the housing can include a top portion, andmechanical biasing elements for mechanically biasing the plurality offluid transmitting vesicles into sealing contact with the housing bottomportion. In one particular embodiment, each fluid transmitting vesiclehas a proximal end portion that includes a flange, and further includesa seal element disposed between the flange and an inner surface of thehousing bottom portion for forming a seal between the interior volumeand an external environment. The biasing elements can be mechanical andcan include a plurality of spring elements each of which are coupled atone end to the proximal end of each the plurality of fluid transmittingvesicles, and at another end to an inner surface of the housing topportion. The springs can apply a mechanical biasing force to the vesicleproximal end to form the seal.

[0023] In a further embodiment, the housing further includes a topportion, and securing element for securing the housing top portion tothe housing bottom portion. The securing element can comprise aplurality of fastener- receiving apertures formed within one of the topand bottom portions of the housing, and a plurality of fasteners formounting within the apertures for securing together the housing top andbottom portions.

[0024] In one embodiment the dispensing element can comprise a pressuresource fluidly coupled to the interior volume of the housing fordisposing the interior volume at a selected pressure condition.Moreover, in an embodiment wherein the fluid transmitting vesicles arefilled through capillary action, the dispensing element can include apressure controller than can vary the pressure source to dispose theinterior volume of the housing at varying pressure conditions. Thisallows the controller varying element to dispose the interior volume ata selected pressure condition sufficient to offset the capillary actionto fill the fluid holding chamber of each vesicle to a predeterminedheight corresponding to a predetermined fluid amount. Additionally, thecontroller can further include a fluid selection element for selectivelydischarging a selected nanovolume fluid amount from the chamber of eachthe vesicle. In one particular embodiment, the apparatus includes apressure controller that operates under the controller of a computerprogram operating on a data processing system to provide variablecontrol over the pressure applied to the interior chamber of thehousing.

[0025] In one embodiment the fluid transmitting vesicle can have aproximal end that opens onto the interior volume of the housing, and thefluid holding chamber of the vesicles are sized to substantiallycompletely fill with the fluid through capillary action without forminga meniscus at the proximal open end. Optionally, the apparatus can haveplural vesicles, wherein a first portion of the plural vesicles includefluid holding chambers of a first size and a second portion includingfluid holding chambers of a second size, whereby plural fluid volumescan be dispensed.

[0026] In another embodiment the apparatus can include, a fluidselection element that has a pressure source coupled to the housing andin communication with the interior volume for disposing the interiorvolume at a selected pressure condition, and an adjustment element thatcouples to the pressure source for varying the pressure within theinterior volume of the housing to apply a positive pressure in the fluidchamber of each the fluid transmitting vesicle to vary the amount offluid dispensed therefrom. The selection element and adjustment elementcan be computer programs operating on a data processing system thatdirects the operation of a pressure controller connected to the interiorchamber.

[0027] In a further alternative embodiment, an apparatus for dispensinga fluid in chemical or biological procedures into one or more wells of amulti-well substrate is provided. The apparatus can include a housinghaving a plurality of sides and a bottom portion having formed therein aplurality of apertures, the walls and bottom portion defining aninterior volume, a plurality of fluid transmitting vesicles, mountedwithin the apertures, having a fluid holding chamber disposed incommunication with the interior volume of the housing, and a fluidselection and dispensing means in communication with the interior volumeof the housing for variably selecting am amount of the fluid loadedwithin the fluid holding chambers of the vesicles to be dispensed from asingle set of the plurality of fluid transmitting vesicles. Accordingly,the dispensing means dispenses a selected amount of the fluid into thewells of the multi-well substrate when the apparatus is disposed overand in registration with the substrate.

[0028] In yet another embodiment, a fluid dispensing apparatus fordispensing fluid in chemical or biological procedures into one or morewells of a multi-well substrate, that includes a housing having aplurality of sides and top and bottom portions, the bottom portionhaving formed therein a plurality of apertures, the walls and top andbottom portions of the housing defining an interior volume, a pluralityof fluid transmitting vesicles, mounted within the apertures, having afluid holding chamber sized to hold nanovolumes of the fluid, the fluidholding chamber being disposed in fluid communication with the volume ofthe housing, and mechanical biasing element for mechanically biasing theplurality of fluid transmitting vesicles into sealing contact with thehousing bottom portion is provided.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029]FIG. 1 illustrates one system provided herein for preparing arraysof a sample material for analysis;

[0030]FIG. 2 illustrates a pin assembly suitable for use with the systemdepicted in FIG. 1 for implementing a parallel process of dispensingmaterial to a surface of a substrate;

[0031]FIG. 3 depicts a bottom portion of the assembly shown in FIG. 2;

[0032]FIG. 4 depicts an alternative view of the bottom portion of thepin assembly depicted in FIG. 2;

[0033] FIGS. 5A-5D depict one method provided herein for preparing anarray of sample material;

[0034] FIGS. 6A-6B depict an alternative assembly for dispensingmaterial to the surface of a substrate.

[0035]FIG. 7 depicts one embodiment of a substrate having wells etchedtherein that are suitable for receiving material for analysis.

[0036]FIG. 8 depicts one example of spectra obtained from a linear timeof flight mass spectrometer instrument and representative of thematerial composition of the sample material on the surface of thesubstrate depicted in FIG. 7; and

[0037]FIG. 9 depicts molecular weights determined for the samplematerial having spectra identified in FIG. 8.

DETAILED DESCRIPTION OF THE ILLUSTRATED EMBODIMENTS

[0038]FIG. 1 illustrates one system provided herein for preparing arraysof sample material for analysis by a diagnostic tool. FIG. 1 depicts asystem that includes a data processor 12, a motion controller 14, arobotic arm assembly 1 6, a monitor element 18A, a central processingunit 18B, a microliter plate of source material 20, a stage housing 22,a robotic arm 24, a stage 26, a pressure controller 28, a conduit 30, amounting assembly 32, a pin assembly 38, and substrate elements 34. Inthe view shown by FIG. 1, it is also illustrated that the roboticassembly 16 can include a moveable mount element 40 and a horizontalslide groove 42. The robotic arm 24 can optionally pivot about a pin 36to increase the travel range of the arm 24 so that arm 24 can disposesthe pin assembly 38 above the source plate 20.

[0039] The data processor 12 depicted in FIG. 1 can be a conventionaldigital data processing system such as an IBM PC compatible computersystem that is suitable for processing data and for executing programinstructions that will provide information for controlling the movementand operation of the robotic assembly 16. It will be apparent to oneskilled in the art that the data processor unit 12 can be any type ofsystem suitable for processing a program of instructions signals thatwill operate the robotic assembly that is integrated into the robotichousing 16. Optionally the data processor 12 can be a micro-controlledassembly that is integrated into robotic housing 16. In furtheralternative embodiments, the system 10 need not be programmable and canbe a singleboard computer having a firmware memory for storinginstructions for operating the robotic assembly 16.

[0040] In the embodiment depicted in FIG. 1, there is a controller 14that electronically couples between the data processor 12 and therobotic assembly 16. The depicted controller 14 is a motion controllerthat drives the motor elements of the robotic assembly 16 forpositioning the robotic arm 24 at a selected location. Additionally, thecontroller 14 can provide instructions to the robotic assembly 16 todirect the pressure controller 28 to control the volume of fluid ejectedfrom the individual pin elements of the depicted pin assembly 38. Thedesign and construction of the depicted motion controller 14 followsfrom principles well known in the art of electrical engineering, and anycontroller element suitable for driving the robotic assembly 16 can beused.

[0041] The robotic assembly 16 depicted in FIG. 1 electronically couplesto the controller 14. The depicted robotic assembly 16 is a gantrysystem that includes an XY table for moving the robotic arm about a XYplane, and further includes a Z axis actuator for moving the robotic armorthogonally to that XY plane. The robotic assembly 16 depicted in FIG.1 includes an arm 24 that mounts to the XY stage which moves the armwithin a plane defined by the XY access. In the depicted embodiment, theXY table is mounted to the Z actuator to move the entire table along theZ axis orthogonal to the XY plane. In this way, the robotic assemblyprovides three degrees of freedom that allows the pin assembly 38 to bedisposed to any location above the substrates 34 and the source plate 20which are shown in FIG. 1 as sitting on the stage 26 mounted to therobotic assembly 16.

[0042] The depicted robotic assembly 16 follows from principles wellknown in the art of electrical engineering and is just one example of arobotic assembly suitable for moving a pin assembly to locationsadjacent a substrate and source plate such as the depicted substrate 34.Accordingly, it will be apparent to one of skill in the art thatalternative robotic systems can be used.

[0043]FIG. 1 depicts an embodiment of a robotic assembly 16 thatincludes a pressure controller 28 that connects via a conduit 30 to themount 32 that connects to the pin assembly 38. In this embodiment themount 32 has an interior channel for fluidicly coupling the conduit 30to the pin assembly 38. Accordingly, the pressure controller 28 isfluidicly coupled by the conduit 30 and the mount 32 to the pin assembly38. In this way the controller 1 4 can send signals to the pressurecontroller 28 to control selectively a fluid pressure delivered to thepin assembly 38.

[0044]FIG. 2 depicts one embodiment of a pin assembly 50 suitable forpractice with the system depicted in FIG. 1 which includes the pressurecontroller 28. In the depicted embodiment, the pin assembly 50 includesa housing formed from an upper portion 52 and a lower portion 54 thatare joined together by the crews 56A and 56B to define an interiorchamber volume 58. FIG. 2 further depicts that to fluidicly seal theinterior chamber volume 58 the housing can include a seal elementdepicted in FIG. 2 as an O-ring gasket 60 that sites between the upperblock and the lower block 54 and surrounds completely the perimeter ofthe interior chamber volume 58. FIG. 2 further depicts that the pinassembly 50 includes a plurality of vesicles 62A-62D, each of whichinclude an axial bore extending therethrough to form the depictedholding chambers 64A-64D. Each of the depicted vesicles extends througha respective aperture 68A-68D disposed within the lower block 54 of thehousing.

[0045] As further shown in the depicted embodiment, each of the vesicles62A-62D has an upper flange portion that sits against a seal element70A-70D to form a fluid-tight seal between the vesicle and the lowerblock 54 to prevent fluid from passing through the apertures 68A-68D. Tokeep the seal tight, the depicted pin assembly 50 further includes a setof biasing elements 74A-74D depicted in FIG. 2 as springs which, in thedepicted embodiments, are in a compressed state to force the flangeelement of the vesicles 62A-62D against their respective seal elements70A-70D. As shown in FIG. 2, the biasing elements 74A-74D extend betweenthe vesicles and the upper block 52. Each of the springs 74A-74D can befixedly mounted to a mounting pad 76A-76D where the spring elements canattach to the upper block 52. The upper block 52 further includes anaperture 78 depicted in FIG. 2 as a centrally disposed aperture thatincludes a threaded bore for receiving a swagelok 80 that can berotatably mounted within the aperture 78.

[0046] As further depicted in FIG. 2, the swagelok 80 attaches by aconduit to a valve 82 than can connect the swagelok 80 to a conduit 84that can be coupled to a pressure source, or alternatively can couplethe swagelok 80 to a conduit 86 that provides for venting of theinterior chamber 58. A central bore 88 extends through the swagelok 80and couples to the tubing element which further connects to the valve 82to thereby fluidicly and selectively couple the interior chamber volume58 to either a pressure source, or a venting outlet.

[0047] The pin assembly 50 described above and depicted in FIG. 2disposed above a substrate element 90 that includes a plurality of wells92 that are etched into the upper surface of the substrate 90. Asillustrated by FIG. 2, the pitch of the vesicles 62A-62D is such thateach vesicle is spaced from the adjacent vesicles by a distance that isan integral multiple of the pitch distance between wells 92 etched intothe upper surface of the substrate 90. As will be seen from thefollowing description, this spacing facilitates the parallel dispensingof fluid, such that fluid can be dispensed into a plurality of wells ina single operation. Each of the vesicles can be made from stainlesssteel, silica, polymeric material or any other material suitable forholding fluid sample. In one example, 16 vesicles are employed in theassembly, which are made of hardened beryllium copper, gold plated overnickel plate. They are 43.2 mm long and the shaft of the vesicle isgraduated to 0.46 mm outer diameter with a concave tip. Such a pin waschosen since the pointing accuracy (distance between the center ofadjacent tips) can be approximately 501 micrometers. However, it will beapparent that any suitable pin style can be employed for-the device,including but not limited to flat, star-shaped, concave, pointed solid,pointed semi-hollow, angled on one or both sides, or other suchgeometries.

[0048]FIG. 3 shows from a side perspective the lower block 54 of the pinassembly 50 depicted in FIG. 2. FIG. 3 shows approximate dimensions forone pin assembly suited for use in the methods and with the apparatusprovided herein. As shown, the lower block 54 has a bottom plate 98 anda surrounding shoulder 100. The bottom plate 98 is approximately 3 mm inthickness and the shoulder 100 is approximately 5 mm in thickness.

[0049]FIG. 4 shows from an overhead perspective the general structureand dimensions for one lower block 54 suitable for use with the pinassembly for use with the pin assembly 50 shown in FIG. 2. As shown inFIG. 4, the lower block 54 includes a four-by-four matrix of apertures68 to provide 16 apertures each suitable for receiving a vesicle. Asdescribed above with reference to FIG. 2, the spacing between theaperture 68 is typically an integral multiple of the distance betweenwells on a substrate surface as well as the wells of a source plate.Accordingly, a pin assembly having the lower block 54 as depicted inFIG. 4 can dispense fluid in up to 16 wells simultaneously. FIG. 4 alsoshows general dimensions of one lower block 54 such that each side ofblock 54 is generally 22 mm in length and the pitch between aperture 68is approximately 4.5 mm. Such a pitch is suitable for use with asubstrate where fluid is to be dispensed at locations approximately 500μm apart, as exemplified by the substrate 90 of FIG. 2. FIG. 4 alsoshows that the lower block 54 can include an optional O-ring groove 94adapted for receiving an O-ring seal element, such as the seal element60 depicted in FIG. 2. It is understood that such a groove element 94can enhance and improve the fluid seal formed by the seal element 60.

[0050] The pinblock can be manufactured of stainless steel as thismaterial can be drilled accurately to about +25 μm, but a variety ofprobe materials can also be used, such as G10 laminate, PMMA or othersuitable material. The pin block can contain any number of apertures andis shown with 16 receptacles which hold the 16 pins in place. Toincrease the pointing accuracy of each pin, an optional alignment placecan be placed below the block so that about 6 mm of the pin tip is leftexposed to enable dipping into the wells of a microtiter plate. Thelayout of the probes in the depicted tool is designed to coordinate witha 384-well microtiter plate, thus the center-to-center spacing of theprobes in 4.5 mm. An array of 4×4 probes was chosen since it wouldproduce an array that would fit in less than one square inch, which isthe travel range of an xy stage of a MALDI TOF MS employed by theassignee. The pintool assembly is completed with a stainless steel coveron the top side of the device which is then attached onto the Z-arm ofthe robot.

[0051] With references to FIG. 5, the operation of one embodiment can beexplained. In this exemplary embodiment, the robotic assembly 16 employsa pin tool assembly 38 that is configured similarly as the pin toolassembly 50 depicted in FIG. 2. The pressure controller 28 selectivelycontrols the pressure within chamber 58. With this embodiment, a controlprogram operates on the data processor 12 to control the roboticassembly 16 in a way that the assembly 16 prints an array of elements onthe substrates 34.

[0052] In a first step, FIG. 5A, the program can direct the roboticassembly 16 to move the pin assembly 38 to be disposed above the sourceplate 20. The robotic assembly 16 will then dip the pin assembly intothe source plate 20 which can be a 384 well DNA source plate. As shownin FIG. 4 the pin assembly can include 16 different pins such that thepin assembly 50 will dip 16 pins into different 16 wells of the 384 wellDNA source plate 20. Next the data processor 12 will direct the motioncontroller 14 to operate the robotic assembly 16 to move the pinassembly to a position above the surface of the substrate 34. Thesubstrate 34 can be any substrate suitable for receiving a sample ofmaterial and can be formed of silicon, plastic, metal, or any other suchsuitable material. Optionally the substrate will have a flat surface,but can alternatively include a pitted surface, a surface etched withwells or any other suitable surface typography. The program operating ondata processor 12 can then direct the robotic assembly, through themotion controller 14, to direct the pressure controller 28 to generate apositive pressure within the interior chamber volume 58. In thispractice, the positive interior pressure will force fluid from theholding chambers of vesicles 62 to eject fluid from the vesicles andinto a respective well 92 of the substrate 90.

[0053] In this practice of the methods and using the apparatus providedherein, the program operating on data processor 12 can also direct thecontroller 14 to control the pressure controller 28 to control fillingthe holding chambers with source material from the source plate 20. Thepressure controller 28 can generate a negative pressure within theinterior chamber volume 58 of the pin assembly. This will cause fluid tobe drawn up into the holding chambers of the vesicles 62A-62D. Thepressure controller 28 can regulate the pressure either by open-loop orclosed-loop control to avoid having fluid overdrawn through the holdingchambers and spilled into the interior chamber volume 58. Loop controlsystems for controlling pressure are well known in the art and anysuitable controller can be employed. Such spillage could causecross-contamination, particularly if the source material drawn from thesource plate 20 varies from well to well.

[0054] In an alternative embodiment, each of the holding chambers64A-64D is sufficiently small to allow the chambers to be filled bycapillary action. In such a practice, the pin assembly can include anarray of narrow bore needles, such as stainless steel needles, thatextend through the apertures of the lower block 54. The needles that aredipped into source solutions will be filled by capillary action. In oneembodiment, the length of capillary which is to be filled at atmosphericpressure is determined approximately by: $H = \frac{2\gamma}{PGR}$

[0055] where H equals Height, gamma equals surface tension, P equalssolution density, G equals gravitational force and R equals needleradius. Thus the volume of fluid held by each vesicle can be controlledby selecting the dimensions of the interior bore. It is understood thatat room temperature water will fill a 15 cm length of 100 μm radiuscapillary. Thus, a short bore nanoliter volume needle will fill to fullcapacity, but should not overflow because the capillary force isunderstood to be too small to form a meniscus at the top of the needleorifice. This prevents cross-contamination due to spillage. In oneembodiment, the vesicles of the pin assembly can be provided withdifferent sized interior chambers for holding and dispensing differentvolumes of fluid.

[0056] In an alternative practice, to decrease the volume of liquid thatis drawn into the holding chambers of the vesicles, a small positivepressure can be provided within the interior chamber volume 58 by thepressure controller 28. The downward force created by the positivepressure can be used to counter the upward capillary force. In this way,the volume of fluid that is drawn by capillary force into the holdingchambers of the vesicles can be controlled.

[0057]FIG. 5B, shows that fluid within the holding chambers of theneedle can be dispensed by a small positive pressure introduced throughthe central bore 88 extending through a swagelok 80. By regulating thepressure pulse that is introduced into the interior chamber volume 58,fluid can be ejected either as a spray or by droplet formation at theneedle tip. It is understood that the rate of dispensing, droplet versusspray, depends in part upon the pressure applied by the pressurecontroller 28. In one practice, pressure is applied in the range ofbetween 10 and 1,000 Torr of atmospheric pressure.

[0058] To this end the data processor 12 can run a computer program thatcontrols and regulates the volume of fluid dispensed. The program candirect the controller 28 to eject a defined volume of fluid, either bygenerating a spray or by forming a drop that sits at the end of thevesicle, and can be contacted with the substrate surface for dispensingthe fluid thereto.

[0059]FIGS. 5C and 5D show the earlier steps shown in FIGS. 5A-5B canagain be performed, this time at a position on the substrate surfacethat is offset from the earlier position. In the depicted process, thepin tool is offset by a distance equal to the distance between two wells92. However, it will be apparent that other offset printing techniquescan be employed.

[0060] It will be understood that several advantages of the pin assemblydepicted in FIG. 2 are achieved. For example, rinsing between dispensingevents is straightforward, requiring only single or multiple pinfillings and emptying events with a rinse solution. Moreover, since allholding chambers fill to full capacity, the accuracy of the volumesdispensed varies only according to needle inner dimensions which can becarefully controlled during pin production. Further the device is costeffective, with the greatest expense attributed to the needles, howeverbecause no contact with a surface is required, the needles are exposedto little physical strain or stress, making replacement rare andproviding long life.

[0061] Alternatively, deposition of sample material onto substratesurface can include techniques that employ pin tool assemblies that havesolid pin elements extending from a block wherein a robotic assemblydips the solid pin elements of the pin assembly into a source of samplematerial to wet the distal ends of the pins with the sample materials.Subsequently the robotic assembly can move the pin assembly to alocation above the substrate and then lower the pin assembly against thesurface of the substrate to contact the individual wetted pins againstthe surface for spotting material of the substrate surface.

[0062]FIGS. 6A and 6B depict another alternative system for dispensingmaterial on or to the surface of the substrate. In particular, FIG. 6Adepicts a jet printing device 110 which includes a capillary element112, a transducer element 114 and orifice (not shown) 118, a fluidconduit 122, and a mount 124 connecting to a robotic arm assembly, suchas the robotic arm 24 depicted in FIG. 1. As further shown in FIG. 6Athe jet assembly 110 is suitable for ejecting from the orifice 118 aseries of drops 120 of a sample material for dispensing sample materialonto the surface 128.

[0063] The capillary 112 of the jet assembly 110 can be a glasscapillary, a plastic capillary, or any other suitable housing that cancarry a fluid sample and that will allow the fluid sample to be ejectedby the action of a transducer element, such as the transducer element114. The transducer element 114 depicted in FIG. 6A is a piezo electrictransducer element which forms around the parameter of the capillary 112and can transform an electrical pulse received from the pulse generatorwithin a robotic assembly 16 to cause fluid to eject from the orifice118 of the capillary 112. One such jet assembly having a piezoelectrictransducer element is manufactured by MicroFab Technology, Inc., ofGermany. Any jet assembly that is suitable for dispensing defined andcontrolled the volumes of fluid can be used, including those that usepiezoelectric transducers, electric transducers, electrorestrictivetransducers, magnetorestrictive transducers, electromechanicaltransducers, or any other suitable transducer element. In the depictedembodiment, the capillary 112 has a fluid conduit 122 for receivingfluid material. In an optional embodiment, fluid can be drawn into thecapillary by action of a vacuum pressure that will draw fluid throughthe orifice 118 when the orifice 118 is submerged in a source of fluidmaterial. Other embodiments of the jet assembly 110 can be employed.

[0064]FIG. 6B illustrates a further alternative assembly suitable foruse herein, and suitable for being carried on the robotic arm of arobotic assembly, such as the assembly 1 6 depicted in FIG. 1. FIG. 6Billustrates four jet assemblies connected together, 130A-130D. Similarto the pin assembly in FIG. 2, the jet assembly depicted in FIG. 6B canbe employed for the parallel dispensing of fluid material. It will beunderstood by the skilled artisan in the art of electrical engineering,that each of the jet assemblies 130A-130D can be operated independentlyof the others, for allowing the selective dispensing of fluid fromselect ones of the jet assemblies. Moreover, each of the jet assemblies130A-130D can be independently controlled to select the volume of fluidthat is dispensed from each respected one of the assembly 130A-130D.Other modifications and alterations can be made to the assembly depictedin FIG. 6B.

[0065] In another aspect, methods for rapidly analyzing sample materialsare provided. To this end sample arrays can be formed on a substratesurface according to any of the techniques discussed above. The samplearrays are then analyzed by mass spectrometry to collect spectra datathat is representative of the composition of the samples in the array.It is understood that the above methods provide processes that allow forrapidly dispensing definite and controlled volumes of analyte material.In particular these processes allow for dispensing sub to low nanolitervolumes of fluid. These low volume deposition techniques generate samplearrays well suited for analysis by mass spectrometry. For example, thelow volumes yield reproducibility of spot characteristics, such asevaporation rates and reduced dependence on atmospheric conditions suchas ambient temperature and light.

[0066] Continuing with the example showing in FIG. 1, the arrays can beprepared by loading oligonucleotides (0.1-50 ng/lll) of differentsequences or concentrations into the wells of a 96 well microtitersource plate 20; the first well can be reserved for holding a matrixsolution. A substrate 34, such as a pitted silicon chip substrate, canbe placed on the stage 26 of the robotics assembly 16 and can be alignedmanually to orient the matrix of wells about a set of reference axes.The control program executing on the data processor 12 can receive thecoordinates of the first well of the source plate 20. The robotic arm 24can dip the pin assembly 38 into source plate 20 such that each of the16 pins is dipped into one of the wells. Each vesicle can fill bycapillary action so that the full volume of the holding chamber containsfluid. Optionally, the program executing on the data processor 12 candirect the pressure controller to fill the interior chamber 58 of thepin assembly 38 with a positive bias pressure that will counteract, inpart, the force of the capillary action to limit or reduce the volume offluid that is drawn into the holding chamber.

[0067] Optionally, the pin assembly 38 can be dipped into the same 16wells of the source plate 20 and spotted on a second target substrate.This cycle can be repeated on as many target substrates as desired. Nextthe robotic arm 24 can dip the pin assembly 38 in a washing solution,and then dip the pin assembly into 16 different wells of the sourceplate 20, and spot onto the substrate target offset a distance from theinitial set of 16 spots. Again this can be repeated for as many targetsubstrates as desired. The entire cycle can be repeated to make a 2×2array from each vesicle to produce an 8×8 array of spots (2×2elements/vesicle×16 vesicles=64 total elements spotted). However, itwill be apparent to one of skill in the art that any process suitablefor forming arrays can be used with the methods herein.

[0068] In an alternative embodiment, oligonucleotides of differentsequences or concentrations can be loaded into the wells of up to threedifferent 384-well microtiter source plates; one set of 16 wells can bereserved for matrix solution. The wells of two plates are filled withwashing solution. Five microtiter plates can be loaded onto the stage ofthe robotic assembly 16. A plurality of target substrates can be placedabutting an optional set of banking or registration pins disposed on thestage 26 and provided for aligning the target substrates along a set ofreference axes. If the matrix and oligonucleotide are not pre-mixed, thepin assembly can be employed to first spot matrix solution on alldesired target substrates. In a subsequent step the oligonucleotidesolution can be spotted in the same pattern as the matrix material tore-dissolve the matrix. Alternatively, a sample array can be made byplacing the oligonucleotide solution on the wafer first, followed by thematrix solution, or by pre-mixing the matrix and oligonucleotidesolutions.

[0069] After depositing the sample arrays onto the surface of thesubstrate, the arrays can be analyzed using any of a variety of means(e.g., spectrometric techniques, such as UV/VIS, IR, fluorescence,chemiluminescence, NMR spectrometry or mass spectrometry. For example,subsequent to either dispensing process, sample loaded substrates can beplaced onto a MALDI-TOF source plate and held there with a set ofbeveled screw mounted polycarbonate supports. In one practice, the platecan be transferred on the end of a probe to be held onto a 1 μmresolution, 1″ travel xy stage (Newport) in the source region of atime-of-flight mass spectrometer. It will be apparent to one of skill inthe art that any suitable mass spectrometry tool can be employed in themethods and with the apparatus and systems provided herein.

[0070] Preferred mass spectrometer formats include, but are not limitedto, ionization (I) techniques including but not limited to matrixassisted laser desorption (MALDI), continuous or pulsed electrospray(ESI) and related methods (e.g. lonspray or Thermospray), or massivecluster impact (MCI); those ion sources can be matched with detectionformats including linear or non-linear reflectron time-of-flight (TOF),single or multiple quadruple, single or multiple magnetic sector,Fourier Transform ion cyclotron resonance (FTICR), ion trap, andcombinations thereof (e.g., ion-trap/time-of-flight). For ionization,numerous matrix/wavelength combinations (MALDI) or solvent combinations(ESI) can be employed. Subattomole levels of protein have been detectedfor example, using ESI (Valaskovic, G. A. et al., (1996) Science 273:1199-1202) or MALDI (Li, L. et al., (1996) J. Am. Chem. Soc 118:1662-1663) mass spectrometry.

[0071] Thus, that in the processes provided herein a completely non-contact, high-pressure spray or partial-contact, low pressure dropletformation mode can be employed. In the latter, the only contact thatwill occur is between the droplet and the walls of the well or ahydrophilic flat surface of the substrate 34. However, in neitherpractice need there be any contact between the needle tip and thesurface.

[0072] Definitions

[0073] As used herein the following terms and phrases shall have themeanings set forth below:

[0074] As used herein, the term “nucleic acid” refers tooligonucleotides or polynucleotides such as deoxyribonucleic acid DNA)and ribonucleic acid (RNA) as well as analogs of either RNA or DNA, forexample made from nucleotide analog, any of which are in single ordouble stranded form, Nucleic acid molecules can by synthetic or can beisolated from a particular biological sample using any of a number orprocedures which are well-known in the art, the particular isolationprocedure chosen being appropriate for the particular biological sample.For example, freeze-thaw and alkaline lysis procedures can be useful forobtaining nucleic acid molecules from solid materials; heat and alkalinelysis procedures can be useful for obtaining nucleic acid molecules forurine; and proteinase K extraction can be used to obtain nucleic acidfrom blood (Rolff, A. et al. PCR: Clinical Diagnostics and Research,Springer (1994)).

[0075] The terms “protein”, “polypeptide” and “peptide” are usedinterchangeably herein when referring to a translated nucleic acid (e.g.a gene product).

[0076] “Sample” as used herein, shall refer to a composition containinga material to be detected. In a preferred embodiment, the sample is a“biological sample” (i.e., any material obtained from a living source(e.g. human, animal, plant, bacteria, fungi, protist, virus). Thebiological sample can be in any form, including solid materials (e.g.tissue, cell pellets and biopsies) and biological fluids (e.g. urine,blood, saliva, amniotic fluid and mouth wash (containing buccal cells)).Preferably solid materials are mixed with a fluid.

[0077] “Substrate” shall mean an insoluble support onto which a sampleis deposited. Examples of appropriate substrates include beads (e.g.,silica gel, controlled pore glass, magnetic, Sephadex/Sepharose,cellulose), capillaries, flat supports such as glass fiber filters,glass surfaces, metal surfaces (steel, gold, silver, aluminum, copperand silicon), plastic materials including multiwell plates or membranes(e.g., of polyethylene, polypropylene, polyamide,polyvinylidenedifluoride), pins (e.g., arrays of pins suitable forcombinatorial synthesis or analysis or beads in pits of flat surfacessuch as wafers (e.g., silicon wafers) with or without plates.

EXAMPLES

[0078] Robot-driven serial and parallel pL-nL dispensing tools were usedto generate 10-10³ element DNA arrays on <1″ square chips with flat orgeometrically altered (e.g. with wells) surfaces for matrix assistedlaser desorption ionization mass spectrometry analysis. In the former, a‘piezoelectric pipette’ (70 μm id capillary) dispenses single ormultiple-0.2 nL droplets of matrix, and then analyte, onto the chip;spectra from as low as 0.2 fmol of a 36-mer DNA have been acquired usingthis procedure. Despite the fast (<5 sec) evaporation, micro-crystals of3-hydroxypicolinic acid matrix containing the analyte are routinelyproduced resulting in higher reproducibility than routinely obtainedwith larger volume preparations; all of 1 00 five fmol sports of a23-mer in 800 μm wells yielded easily interpreted mass spectra, with99/100 parent ion signals having signal to noise ration of >5. In asecond approach, probes from 384 well microtiter plate are dispensed 1 6at a time into chip wells or onto flat surfaces using an array of springloaded pins which transfer-20 nL to the chip by surface contact; MSanalysis of array elements deposited with the parallel method arecomparable in terms of sensitivity and resolution to those made with theserial method.

[0079] I. Description of the Piezoelectric Serial Dispenser.

[0080] The experimental system built on a system purchased fromMicrodrop GmbH, Norderstedt Germany and includes a piezoelectric elementdriver which sends a pulsed signal to a piezoelectric element bonded toand surrounding a glass capillary which holds the solution to bedispensed; a pressure transducer to load (by negative pressure) or empty(by positive pressure) the capillary; a robotic xyz stage and robotdriver to maneuver the capillary for loading, unloading, dispensing, andcleaning, a stroboscope and driver pulsed at the frequency of the piezoelement to enable viewing of ‘suspended’ droplet characteristics;separate stages for source and designation plates or sample targets(i.e. Si chip); a camera mounted to the robotic arm to view loading todesignation plate; and a data station which controls the pressure unit,xyz robot, and piezoelectric driver.

[0081] II. Description of the Parallel Dispenser.

[0082] The robotic pintool includes 16 probes housed in a probe blockand mounted on an X Y, Z robotic stage. The robotic stage was a gantrysystem which enables the placement of sample trays below the arms of therobot. The gantry unit itself is composed of X and Y arms which move 250and 400 mm, respectively, guided by brushless linear servo motors withpositional feedback provided by linear optical encoders. A lead screwdriven Z axis (50 mm vertical travel) is mounted to the xy axis slide ofthe gantry unit and is controlled by an in-line rotary servo motor withpositional feedback by a motor-mounted rotary optical encoder. The workarea of the system is equipped with a slide-out tooling plate that holdsfive microtiter plates (most often, 2 plates of wash solution and 3plates of sample for a maximum of 1152 different oligonucleotidesolutions) and up to ten 20×20 mm wafers. The wafers are placedprecisely in the plate against two banking pins and held secure byvacuum. The entire system is enclosed in plexi-glass housing for safetyand mounted onto a steel support frame for thermal and vibrationaldamping. Motion control is accomplished by employing a commercial motioncontroller which was a 3-axis servo controller and is integrated to acomputer; programming code for specific applications is written asneeded.

[0083] Samples were dispensed with the serial system onto severalsurfaces which served as targets in the MALDI TOF analysis including [1]A flat stainless steel sample target as supplied for routine use in aThermo Bioanalysis Vision 2000; [2] the same design stainless steeltarget with micromachined nonpits; [3] flat silicon (Si) wafers; [4]polished flat Si wafers; [5] Si wafers with rough (3-6 pLm features)pits; [6](a) 12×12 or ((b) 18×18) mm Si chips with (a) 10×10 (or (b)16×16) arrays of chemically etched wells, each 800×8001 lm on a sidewith depths ranging from 99-400 (or(b) 120) micrometer, pitch (a) 1.0(or(b) 1.125 mm); [7] 15×15 mm Si chips with 28×28 arrays of chemicallyetched wells, each 450×450 micrometer on a side with depths ranging from48-300 micrometer, pitch 0.5 mm; [8]flat polycarbonate or otherplastics; [9] gold and other metals; [10] membranes; [11] plasticsurfaces sputtered with gold or other conducting materials. Thedispensed volume is controlled from 10⁻¹⁰ to 10⁻⁶ L by adjusting thenumber of droplets dispensed.

[0084] Sample Preparation and Dispensing: Serial Oligonucleotides(0.1-50 ng/microliter of different sequence or concentrations wereloaded into wells of a 96 well microtiter plate; the first well wasreserved for matrix solution. A pitted chip (target 6 a in MALDItargets' section) was placed on the stage and aligned manually. Into the(Windows-based) robot control software were entered the coordinates ofthe first well, the array size (ie number of spots in x and y) andspacing between elements, and the number of 0.2 nL drops per arrayelement. The capillary was filled with ˜10 μl rinse H₂O, automaticallymoved in view of a strobe light-illuminated camera for checking tipintegrity and cleanliness while in continuous pulse mode, and emptied.The capillary was then filled with matrix solution, again checked at thestroboscope, and then used to spot an array onto flat or pittedsurfaces. For reproducibilty studies in different MS modes, typically a101×10 array of 0.2-20 nL droplets were dispensed. The capillary wasemptied by application of positive pressure, optionally rinsed with H₂O,and let to the source oligo plate where ˜5 μL of 0.05-2.0 μM syntheticoligo were drawn. The capillary was then rastered in a series over eachof the matrix spots with 0.2-20 nL aqueous solution added to each.

[0085] Sample Preparation and Dispensing:

[0086] Parallel Programs were written to control array making by offsetprinting; to make an array of 64 elements on 10 wafers, for example, thetool was dipped into 16 wells of a 384 well DNA source plate, moved tothe target (e.g. Si, plastic, metal), and the sample spotted by surfacecontact. The tool was then dipped into the same 16 wells and spotted onthe second target; this cycle was repeated on all ten wafers. Next thetool was dipped in washing solution, then dipped into 16 different wellsof the source plate, and spotted onto the target 2.25 mm offset from theinitial set of 16 spots; again this as repeated on all 10 wafers; theentire cycle was repeated to make a 2×2 array from each pin to producean 8×8 array of spots (2×2 elements/pin X 16 pins=64 total elementsspotted).

[0087] To make arrays for MS analysis, olegonucleotides of differentsequences or concentrations were loaded into the wells of up to threedifferent 384-well microtiter plates, one set of 16 wells was reservedfor matrix solution. The wells of two plates were filled with washingsolution. The five microtiter plates were loaded onto the slide-outtooling plate. Ten wafers were placed abutting the banking pins on thetooling plate, and the vacuum turned on. In cases where matrix andoligonucleotide were not pre-mixed, the pintool was used to spot matrixsolution first on all desired array elements of the ten wafers. For thisexample, a 16×16 array was created, thus the tool must spot each of theten wafers 16 times, with an offset of 1.125 mm. Next, theoligonucleotide solution was spotted in the same pattern to re-dissolvethe matrix, Similarly, an array could be made by placing theoligonucleotide solution on the wafer first, followed by the matrixsolution, or by pre-mixing the matrix and oligonucleotide solutions.

[0088] Mass spectrometry.

[0089] Subsequent to either dispensing scheme, loaded chips were heldonto a MALDI-TOF source plate with a set of beveled screw mountedpolycarbonated supports. The plate was transferred on the end of a probeto be held onto a 1 μm resolution, 1″ travel xy stage (Newport) in thesource region of a time-of-flight mass spectrometer. The instrument,normally operated with 18-26 kV extraction, could be operated in linearor curved field reflectron mode, and in continuous or delayed extractionmode.

[0090] Observations

[0091] I. Serial dispensing with the piezoelectric pipette. Whiledelivery of a saturated 3HPA solution can result in tip clogging as thesolvent at the capillary-air interface evaporates, pre-mixing DNA andmatrix sufficiently dilutes the matrix such that it remains in solutionwhile stable sprays which could be maintained until the capillary wasemptied were obtained; with 1:1 diluted (in H₂O) matrix solution,continuous spraying for >>10 minutes was possible. Turning off the piezoelement so that the capillary sat inactive for >5 minutes, andreactivating the piezo element also did not result in a cloggedcapillary.

[0092] Initial experiments using stainless steel sample targets asprovided by Finnigan Vision 2000 MALDI-TOF system run in reflectron modeutilized a pre-mixed solution of the matrix and DNA prior to dispensingonto the sample target. In a single microtiter well, 50,uL saturatedmatrix solution, 25 μL of a 51 μL solution of the 12-mer (ATCG)3, and 25μL of a 51 μL solution of the 28-mer (ATCG)7 were mixed. A set of 10×10arrays of 0.6 μL drops was dispensed directly onto a Finnigan Vision2000 sample target disk; MALDI-TOF mass spectrum was obtained from asingle array element which contained 750 attomoles of each of the twooligonucleotides. Interpretable mass spectra has been obtained for DNAsas large as a 53-mer (350 amol loaded, not shown) using this method.

[0093] Mass spectra were also obtained from DNAs microdispensed into thewells of a silicon chip. FIG. 7 shows a 12×12mm silicon chip with 100chemically etched wells; mask dimensions and etch time were set suchthat fustum (i.e., inverted flat top pyramidal) geometry wells with800×800 μm (top surface) and 100 μm depth were obtained. Optionally, thewells can be roughed or pitted. As described above, the chip edge wasaligned against a raised surface on the stage to define the x and ycoordinate systems with respect to the capillary. (Alternatives includeoptical alignment, artificial intelligence pattern recognition routines,and dowel-pin based manual alignment). Into each well was dispensed 20droplets (˜5 nL) of 3-HPA matrix solution without analyte; for the 50%CH₃CN solution employed, evaporation times for each droplet were on theorder of 5-10 seconds. Upon solvent evaporation, each microdispensedmatrix droplet as viewed under a 120× stereomicroscope generallyappeared as an amorphous and ‘milky’ flat disk; such appearances areconsistent with those of droplets from which the FIG. 3b spectrum wasobtained. Upon tip emptying, rinsing, and refilling with a 1.4 μmaqueous solution of a 23-mer DNA (Mr(calc)=6967 Da), the capillary wasdirected above each of the 100 spots of matrix where 5 nL of the aqueousDNA solution was dispensed directly on top of the matrix droplets.Employing visualization via a CCD camera, it appeared that the aqueousanalyte solution mixed with and re-dissolved the matrix (completeevaporation took −10 sec at ambient temperature and humidity). Theamorphous matrix surfaces were converted to true micro-crystallinesurfaces, with crystalline features on the order of <1 μm.

[0094] Consistent with the improved crystallization afforded by thematrix re-dissolving method, mass spectrum acquisition appeared morereproducible than with pre-mixed matrix plus analyte solutions; each ofthe 100 five fmol spots of the 23-mer yielded interpreted mass spectra(FIG. 8), with 99/100 parent ion signals having signal to noise rationsof >5; such reproducibility was also obtained with the flat silicon andmetallic surfaces tried (not shown). The FIG. 8 spectra were obtained ona linear TOF instrument operated at 26 kV. Upon internal calibration ofthe top left spectrum (well ‘k1’) using the singly and doubly chargedmolecular ions, and application of this calibration file to all otherspectra as an external calibration (FIG. 9), a standard deviation of <9Da from the average molecular weight was obtained, corresponding to arelative standard deviation of ˜0.1%.

[0095] II. Parallel Dispensing with robotic pintool. Arrays were madewith offset printing as described above. The velocity of the X and Ystages are 35 inches/sec, and the velocity of the Z stage is 5.5inches/sec. It is possible to move the X and Y stages at maximumvelocity to decrease the cycle times, however the speed of the Z stageis to be decreased prior to surface contact with the wafer to avoiddamaging it. At such axes speeds, the approximate cycle time to spot 16elements (one tool impression of the same solutions) on all ten wafersis 20 seconds, so to make an array of 256 elements would take ˜5.3minutes. When placing different oligonucleotide solutions on the array,an additional washing step much be incorporated to clean the pin tipprior to dipping in another solution, thus the cycle time would increaseto 25 seconds or 6.7 minutes to make 10 wafers.

[0096] Sample delivery by the tool was examined using radio-labeledsolutions and the phosphorimager as described previously; it wasdetermined that each pin delivers approximately 1 nL of liquid. Thespot-to-spot reproducibility is high. An array of 256 oligonucleotideelements of varying sequence and concentration was made on flat siliconwafers using the pintool, and the wafer was analyzed by MALDI-TOF MS.

[0097] It will be understood that the above-described examples andillustrated embodiments are provided for describing the invention setforth herein and are not to be taken as limiting in any way, and thescope of the invention is to understood by the claims.

We claim:
 1. A dispensing apparatus for dispensing nanovolumes of fluidin chemical or biological procedures onto the surface of a substrate,comprising a housing having a plurality of sides and a bottom portionhaving formed therein a plurality of apertures, said walls and bottomportion of said housing defining an interior volume, one or more fluidtransmitting vesicles, mounted within said apertures, having ananovolume sized fluid holding chamber for holding nanovolumes of fluid,said fluid holding chamber being disposed in fluid communication withsaid interior volume of said housing, and dispensing means incommunication with said interior volume of said housing for selectivelydispensing nanovoluments of fluid from said nanovolume sized fluidtransmitting vesicles when the fluid is loaded with said fluid holdingchambers of said vesicles, whereby said dispensing means dispensesnanovolumes of the fluid onto the surface of the substrate when theapparatus is disposed over and in registration with the substrate. 2.The apparatus of claim 1 , wherein each said fluid transmitting vesiclehas an open proximal end and a distal tip portion that extends beyondsaid housing bottom portion when mounted within said apertures, saidopen proximal end disposing said fluid holding chamber in fluidcommunication with said interior volume when mounted with the apertures.3. The apparatus of claim 1 , wherein said plurality of fluidtransmitting vesicles are removably and replaceably mounted within saidapertures of said housing.
 4. The apparatus of claim 1 , wherein saidplurality of fluid transmitting vesicles include a glue seal for fixedlymounting said vesicles within said housing.
 5. The apparatus of claim 1, wherein said fluid holding chamber includes a narrow boredimensionally adapted for being filled with the fluid through capillaryaction.
 6. The apparatus of claim 1 , wherein each said fluid holdingchamber of said plurality of fluid transmitting vesicles are sized tofill substantially completely with the fluid through capillary action.7. The apparatus of claim 1 , wherein said plurality of fluidtransmitting vesicles comprise an array of fluid delivering needles. 8.The apparatus of claim 7 , wherein said fluid delivering needles areformed of metal.
 9. The apparatus of claim 7 , wherein said fluiddelivering needles are formed of glass.
 10. The apparatus of claim 7 ,wherein said fluid delivering needles are formed of silica.
 11. Theapparatus of claim 7 , wherein said fluid delivering needles are formedof polymeric material.
 12. The apparatus of claim 1 , wherein the numberof said plurality of fluid transmitting vesicles is less than or equalto the number of wells of a multi-well substrate.
 13. The apparatus ofclaim 1 , wherein said housing further includes a top portion, anfurther comprising mechanical biasing means of mechanically biasing saidplurality of fluid transmitting vesicles into sealing contact with saidhousing bottom portion.
 14. The apparatus of claim 13 , wherein eachsaid fluid transmitting vesicle has a proximal end portion that includesa flange, and further comprising a sealer element disposed between theflange and an inner surface of the housing bottom portion for forming aseal between the interior volume and an external environment.
 15. Theapparatus of claim 14 , wherein said mechanical biasing means includes aplurality of spring elements each of which are coupled at one end tosaid proximal end of each said plurality of fluid transmitting vesicles,and at another end to an inner surface of said housing top portion, saidspring element applying a mechanical biasing force to said vesicleproximal end to form said seal.
 16. The apparatus of claim 1 , whereinsaid housing further includes a top portion, and further comprisingsecuring means for securing said housing top portion to said housingbottom portion.
 17. The apparatus of claim 16 , wherein said securingmeans comprises a plurality of fastner-receiving apertures formed withinone of said top and bottom portions or said housing, and a plurality offastners for mounting within said apertures for securing together saidhousing top and bottom portions.
 18. The apparatus of claim 1 , whereinsaid dispensing mens comprises a pressure source fluidly coupled to saidinterior volume of said housing for disposing said interior volume at aselected pressure condition.
 19. The apparatus of claim 18 , whereinsaid fluid transmitting vesicles are filled through capillary action,and wherein said dispensing means further comprises means for varyingsaid pressure source to dispose said interior volume of said housing atvarying pressure conditions, said means for varying disposing saidinterior volume at a selected pressure condition sufficient to offsetsaid capillary action to fill the fluid holding chamber of each vesicleto a predetermined height corresponding to a predetermined fluid amount.20. The apparatus of claim 1 9, wherein said means for varying furthercomprises fluid selection means for selectively discharging a selectednanovolume fluid amount from said chamber of each said vesicle.
 21. Theapparatus of claim 1 , wherein said fluid transmitting vesicle has aproximal end that opens onto said interior volume of sid housing, andwherein said fluid holding chamber of said vesicles are sized tosubstantially completely fill with the fluid through capillary actionwithout forming a meniscus at said proximal open end.
 22. The apparatusof claim 1 , wherein said dispensing means comprises fluid selectionmeans for selectively varying the amount of fluid dispensed from saidfluid holding chamber of each vesicle.
 23. The apparatus according toclaim 1 , having plural vesicles, wherein a first portion of said pluralvesicles include fluid holding chambers of a first size and a secondportion including fluid holding chambers of a second size, wherebyplural fluid volumes can be dispensed.
 24. The apparatus of claim 22 ,wherein said fluid selection means comprises a pressure source coupledto said housing and in communications with said interior volume fordisposing said interior volume at a selected pressure condition, andadjustment means coupled to said pressure source for varying saidpressure within said interior volume of said housing to apply a positivepressure in said fluid chamber of each said fluid transmitting vesicleto vary the amount of fluid dispensed therefrom.
 25. A fluid dispensingapparatus for dispensing a fluid in chemical or biological proceduresinto one or more wells of a multi-well substrate, comprising a housinghaving a plurality of sides and a bottom portion having formed therein aplurality of apertures, said walls and bottom portion defining aninterior volume, a plurality of fluid transmitting vesicles, mountedwithin said apertures having a fluid holding chamber disposed incommunication with said interior volume of said housing, a fluidselection and dispensing means in communication with said interiorvolume of said housing for variably selecting an amount of the fluidloaded with said fluid holding chambers of said vesicles to be dispensedfrom a single set of plurality of fluid transmitting vesicles, andwhereby said dispensing means dispenses a selected amount of the fluidinto the wells of the multi-well substrate when the apparatus isdisposed over and in registration with the substrate.
 26. The fluiddispensing apparatus of claim 25 , wherein said fluid selection anddispensing means is adapted to select various amounts of fluid to bedispensed from said single set of vesicles.
 27. The fluid dispensingapparatus of claim 25 , wherein said fluid selection and dispensingmeans comprises a pressure source fluidly coupled to said interiorvolume of said housing for disposing said interior volume at a selectedpressure condition.
 28. The fluid dispensing apparatus of claim 27 ,further compromising means for varying the pressure within the interiorvolume of the housing to select the amount of fluid to dispense fromsaid fluid transmitting vesicles.
 29. The fluid dispensing apparatus ofclaim 27 , wherein said fluid transmitting vesicles are filled with thefluid through capillary action, and further comprising means for varyingsaid pressure source to dispose said interior volume of said housing atvarying pressure conditions, said means for varying disposing saidinterior volume at a pressure condition sufficient to offset saidcapillary action to fill the fluid holding chamber of each vesicle to apredetermined height corresponding to a predetermined fluid amount. 30.The fluid dispensing apparatus of claim 25 , wherein said fluidselection means comprises a pressure source coupled to said housing andin communication with said interior volume for disposing said interiorvolume at a selected pressure condition, and adjustment means coupled tosaid pressure source for varying said pressure within said interiorvolume of said housing to apply a positive pressure in said fluidchamber of each said fluid transmitting vesicle to vary the amount offluid dispensed therefrom.
 31. A fluid dispensing apparatus fordispensing fluid in chemical or biological procedures into one or morewells of a multi-well substrate, said apparatus comprising a housinghaving a plurality of sides and top and bottom portions of said bottomportion having formed therein a plurality of apertures, said walls andtop and bottom portions of said housing defining an interior volume, aplurality of fluid transmitting vesicles, mounted within said apertureshaving a fluid holding chamber sized to hold nanovolumes of the fluid,said fluid holding chamber being disposed in fluid communication withsaid volume of said housing and mechanical biasing means formechanically biasing said plurality of said transmitting vesicles intosealing contact with said housing bottom portion.
 32. The fluiddispensing apparatus of claim 31 , wherein each said fluid transmittingvesicle has a proximal end portion that includes a flange, and furthercomprising a sealer element disposed between the flange and an innersurface of the housing bottom portion for forming a pressure and fluidseal between the internal and external environment.
 33. The fluiddispensing apparatus of claim 31 , wherein said mechanical biasing meansincludes a plurality of spring elements each of which are coupled at oneend to said means includes a plurality of spring elements each of whichare coupled at one end to said proximal end of said fluid transmittingvesicle, and at another end to an inner surface of said housing topportion, said spring elements applying a mechanical biasing force tosaid vesicle proximal end to form said fluid and pressure seal.
 34. Thefluid dispensing apparatus of claim 31 , further comprising securingmeans for securing said housing top portion to said housing bottomportion.
 35. The fluid dispensing apparatus of claim 34 , wherein saidsecuring means comprises a plurality of fastener-receiving aperturesformed within one of said top and bottom portions of said housing, and aplurality of fasteners for mounting within said apertures for securingsaid housing top and bottom portions together.
 36. The fluid dispensingapparatus of claim 31 , further comprising dispensing means incommunication with said interior volume of said housing for selectivelydispensing the fluid from said fluid transmitting vesicles when thefluid is loaded within said fluid holding chambers of said vesicles,whereby said dispensing means dispenses the fluid into the wells of themulti-well substrate when the apparatus is disposed over an inregistration with the substrate.
 37. The fluid dispensing apparatus ofclaim 36 , wherein said dispensing means comprises a pressure sourcefluidly coupled to said interior volume of said housing for disposingsaid interior volume at a selected pressure condition.
 38. The fluiddispensing apparatus of claim 31 , wherein said plurality of fluidtransmitting vesicles are removably and replaceably mounted within saidapertures of said housing.
 39. The fluid dispensing apparatus of claim31 , wherein said plurality of fluid transmitting vesicles comprises anarray of fluid delivering needles.
 40. The fluid dispensing apparatus ofclaim 36 , wherein said fluid transmitting vesicles are filled with thefluid through capillary action, and wherein said dispensing meansfurther comprises means for varying said pressure source to dispose saidinterior volume of said housing at varying pressure conditions, saidmeans for varying disposing said interior volumes at a selected pressurecondition sufficient to offset said capillary action to fill the fluidholding chamber of each vesicle to a predetermined height correspondingto a predetermined fluid amount.
 41. The fluid dispensing apparatus ofclaim 36 , wherein said dispensing means comprises fluid selection meansfor selectively varying the amount of fluid dispensed from said fluidholding chamber of each vesicle.
 42. The fluid dispensing apparatus ofclaim 31 , further comprising a pressure source coupled to the housingand in communication with the interior volume for disposing the interiorvolume at a selected pressure condition, and adjustment means coupled tothe pressure source for varying the pressure within the interior volumeof the housing to apply a positive pressure to the fluid chamber of eachthe fluid transmitting vesicle to vary the amount of fluid dispensedtherefrom.